One-third of the world's population is infected with Mycobacterium tuberculosis (M. tb), the etiologic agent of TB. The World Health Organization estimates that about eight to ten million new TB cases occur annually worldwide and the incidence of TB is currently increasing. Together with malaria and HIV-AIDS, TB is one of the three leading causes of death from a single infectious agent, and approximately two million deaths are attributable to TB annually (WHO Report, 2008). The only currently licensed vaccine is Mycobacterium bovis Bacille Calmette-Guerin (M. bovis BCG), an attenuated strain of M. bovis, which has been administered to over four billion people over the years since 1921, when it was first used. When administered at birth, M. bovis BCG confers consistent and reliable protection against disseminated disease in the first ten years of life (Rodrigues et al., 1993). However, the protection conferred against pulmonary disease in adolescents and adults is much more variable (Colditz et al., 1994). The main current approach in developing improved prophylactic TB vaccines is to use modified M. bovis BCG or M. tb to improve upon M. bovis BCG as a priming vaccine, possibly followed by boosting some time later with selected immunodominant antigens as a protein or viral vector vaccine (Barker et al., 2009; STOP-TB Partnership, 2009).
In the case of tuberculosis, alveolar macrophages successfully phagocytose M. tb and are the primary site of infection. However, the mycobacteria interfere with phagosomal maturation, thus enabling the mycobacteria to partially evade many of the immune mechanisms that would otherwise eliminate them.(50) The bacterial genes and pathways that are essential for the intracellular growth and survival of M. tb are attractive drug targets and several of such bacterial virulence mechanisms have been suggested.(13, 14) However, the functional validation of the bacterial components suspected to be involved in M. tb host interactions requires the generation of mutants. These critical experiments are hampered by the slow growth and poor homologous recombination efficiency of M. tb(39) and by the requirement to work in costly high-biosafety laboratories. The combination of these features makes mutant generation a very lengthy, cumbersome and expensive process. Therefore, many researchers resort to non-pathogenic fast-growing mycobacterial species to generate mutants, with results that are not necessarily to be extrapolated to their slow-growing cousins.(24) The attenuated vaccine strain M. bovis BCG is a more faithful, yet safer model organism for many aspects of M. tb host interactions.(8, 17) It is also slow growing and is genomically extremely similar to M. tuberculosis (>99%), only lacking a small number of genomic regions, of which the RD1 locus has primarily resulted in its attenuation.(2, 33) Each BCG strain has multiple deletions, of which only the RD1 locus is common to all.
It should be noted that the unusual cell wall of mycobacteria is one of the factors that enable the mycobacteria to successfully colonize their host. The cell wall is composed of a complex of lipids, glycolipids and proteins that interact with macrophages.(5) One such cell envelope component is lipoarabinomannan (LAM), which, along with its precursors lipomannan (LM) and phosphatidyl-myo-inositol mannosides (PIMs), is noncovalently anchored to the plasma membrane. LAM contains a mannosylphosphatidyl-myo-inositol anchor (MPI), a mannan core and long, substituted arabinan branches. The mannan backbone consists of α-1,6-linked mannose units with single residue α-1,2-Manp substitutions. The arabinan polymer is composed of α-1,5-linked Araf units and is substituted with branched hexa-arabinofuranosides and linear tetra-arabinofuranosides. The non-reducing termini of these arabinan side-chains terminate in cap motifs. In slow-growing pathogenic strains, these motifs consist of one, two or three α-1,2-linked Manp residues with the dimannoside being the most abundant. In contrast, in non-pathogenic strains, there are usually inositol phosphate caps (PILAM in M. smegmatis) or no caps at all (AraLAM in M. chelonae).(7) ManLAM (mannosylated lipoarabinomannan) has been reported to play a role in subverting host cell signaling pathways(18) and blocking phagosome maturation(15) in host macrophages.
LAM is built from phosphatidyl-myo-inositol (PI), the synthesis of which requires the activity of the diacylglycerol kinase Mb2276.(45) PI undergoes a series of sequential α-mannosylations, catalyzed by PimA (Mb2642c), PimB (Mb0572), PimC (Mb 1785c) and PimF (Mb1538).(25, 26, 38, 57) The glycosyl donor substrate for all mannosylations in ManLAM synthesis from PI is polyprenol phosphomannose, which is synthesized from GDP-mannose by the polyprenol phosphomannose synthase ppm1 (Mb2077c).(19) GDP-mannose synthesis in this process involves the phosphomannose mutase pmmB (Mb3336).(55) LM is synthesized on the PIM4 precursor by the addition of a linear α-1,6-mannose polymer of 21-34 residues. The mannosyltransferase Mb2196 is required for this modification.(23) A previous study showed that Rv2181 (Mb2203) is the mannosyltransferase responsible for the addition of single α-1,2-Manp residues to the mannan core of LM/LAM.(20) In this latter study, the inactivation of the corresponding homologue in M. smegmatis leads to the production of a truncated LAM and the absence of LM. However, more recent data reveal that a similar inactivation in M. tb leads to production of lower molecular weight LAM, but does not affect the synthesis of LM, revealing a role for this mannosyltransferase in the terminal capping of LAM.(24) LAM is derived from LM through the addition of poly α-1,5-araf chains, the synthesis of which requires embC.(4, 65) The transcription of embC is regulated by the serine threonine kinase pknH via the phosphorylation of the transcriptional regulator EmbR.(59) The linear arabinan branches are further substituted with hexa- and tetra-arabinofuranoside structures. These non-reducing arabinan termini of the LAM are capped in M. tuberculosis and M. bovis BCG, to varying degrees, with one to three (α-1,2)-Manp residues. The first mannosyltransferase involved in this α-1,2-mannose capping is encoded by Rv1635c and its homologue in M. bovis BCG, Mb 1661c.(11) While the gene Mb1661c is likely responsible for the addition of the first mannose residue of the cap, the mannosyltransferase Mb2203 is thought to add the additional one or two mannose residues to generate di- and tri-Manp capped LAM.(24) Other factors influencing the virulence of M. tb include the lipoprotein signal peptidase, LspA (Mb1566),(53) the lipid phosphatase SapM (Mb3338),(42) and the Zn metalloprotease Zmp1 (Mb0204c).(35) 
Most work in the tuberculosis vaccine field is directed by the belief that BCG is “missing something of M. tuberculosis” and that this either has to be added back to BCG to improve vaccine-induced protection, or that, conversely, M. tb should be attenuated to the low virulence of BCG while keeping its immunodominant antigens. Examples of the former are recombinant BCG strains overexpressing immunodominant M. tb antigens (Castanon-Arreola et al., 2005; Horwitz & Harth, 2003; Pym et al., 2003) and examples of the latter are virulence factor (Copenhaver et al., 2004), auxotrophic (Sambandamurthy et al., 2005; Sambandamurthy et al., 2002) or signal transduction (Martin et al., 2006) mutants of M. tb. Moreover, improved induction of phagosome maturation and apoptosis in phagocytes (Grode et al., 2005; Hinchey et al., 2007; Sadagopal et al., 2009; Sun et al., 2009; Velmurugan et al., 2007) is pursued to increase cross-presentation and therewith, vaccine efficiency. However, of all the engineered live vaccines, in direct comparisons with BCG vaccine, only five have been demonstrated to be sufficiently promising in experimental animals to be moved forward to Phase I clinical trials (STOP-TB Partnership, 2009): BCG overexpressing antigen 85b (rBCG30 prolonged survival in guinea pigs) (Horwitz & Harth, 2003); the urease C-deficient listeriolysin-secreting recombinant BCG (Grode et al., 2005; Tchilian et al., 2009) (ΔureC hly+ rBCG decreased CFU counts in lungs of mice), the urease C-deficient perfringiolysin-secreting recombinant BCG (Sun et al., 2009) (safer than BCG in SCID mouse study), the phoP mutant of M. tuberculosis (Martin et al., 2006; Verreck et al., 2009) (prolonged survival in guinea pigs, under high-dose challenge, while equal protection against low-dose challenge, and equal protection in mice; decreased CFU counts in lungs of rhesus macaques) and the secA2 mutant of M. tuberculosis (Hinchey et al., 2007; Sadagopal et al., 2009) (prolonged survival in mice). As M. bovis BCG is a live vaccine, there are regulatory and safety concerns toward transgenic strains being implemented at the massive scale, which is relevant for TB protection (e.g., transgene stability; risk for horizontal gene transfer of the transgene between the vaccine and M. tb itself (Krzywinska et al., 2004); different deliberate environmental release GMO regulations throughout the world) (Kamath et al., 2005; Walker et al., 2010). There are also potential safety issues with attenuated M. tb as a vaccine, and special precautions have to be taken (such as double mutations, with each single mutation resulting in sufficient attenuation) (Kamath et al., 2005; Walker et al., 2010). If a M. bovis BCG strain with better protective capability than the licensed strain BCG itself could be found in which the improved protection was brought about by targeted inactivation of endogenous genes rather than expressing heterologous transgenes, these concerns would be reduced and clinical implementation and public acceptance would be more straightforward.